Immunoreactive luteinizing hormone-releasing hormone in the frog (Rana esculenta) brain: distribution pattern in the adult, seasonal changes, castration effects, and developmental aspects

The present work describes the neuroanatomical distribution of the immunoreactive luteinizing hormone-releasing hormone (ir-LHRH) system in the brain of adult male and female, castrated male and developing Rana esculenta. No obvious sex differences in the distribution pattern of ir-LHRH were observed. Immunoreactive neuronal cell bodies are not contained within a single anatomically defined area of the brain. They are present as distinct groups in the olfactory bulbs, medial septal area, anterior preoptic area (APOA), retrochiasmatic area of the infundibulum, and interpeduncular nucleus-tegmentum area. Of the entire brain, the medial septal-APOA region exhibits the highest frequency of ir-LHRH cell bodies in both sexes. ir-LHRH fiber projections are present in the olfactory bulbs, medial septal area, APOA, floor of the diencephalon, subhabenular-periventricular area in the epithalamus, lateral suprachiasmatic area, ventrolateral infundibulum, median eminence, pars nervosa, optic tectum, interpeduncular nucleus-tegmentum area, and rhombencephalon grey. Castration seems to bear no effect on the pattern of ir-LHRH system in the frog brain. The influence of castration consisted in decreased intensity of the immunostaining and frequency of occurrence of the septal-APOA neuronal cell bodies. In median eminence, castration also induced a sensible decrease in the immunoreactivity, whereas in the pars nervosa of 50-day castrates ir-LHRH fibers totally disappeared. During ontogenesis, ir-LHRH elements first become evident in stage 31 tadpoles (beginning of metamorphic climax); LHRH immunoreaction is restricted to the cell bodies and fibers in the APOA and some fibers in the ventral hypothalamus and a few in median eminence. This condition remains unaltered until stage 33 when the tail is almost totally resorbed. The possible implications of the ir-LHRH-containing brain areas in the different aspects of reproduction in the frog are briefly discussed.     

Regulation of androgen metabolism and luteinizing hormone-releasing hormone content in discrete hypothalamic and limbic areas of male rhesus macaques

The conversion of androgens to active metabolites by neural tissue is believed to be an essential component in the cellular mechanism of androgen-induced neuroendocrine responses. In this study, we measured the in vitro aromatization and 5 alpha-reduction of androgens in incubations of microdissected brain regions from four intact and five castrated (6 weeks) adult male rhesus monkeys. Individual nuclei were microdissected from 600-microns frozen brain sections and homogenized in a potassium phosphate buffer. Aromatase activity was measured by a radiometric assay that uses the incorporation of tritium from [1 beta-3H]androstenedione into 3H2O as an index of estrogen formation. We estimated 5 alpha-reductase activity by isolating 5 alpha-dihydrotestosterone on two different chromatography systems and measuring the amount of this product formed from [1 alpha,2 alpha-3H]testosterone. We acidified a portion of each homogenate and determined LHRH content by RIA. Between brain nuclei, aromatase activity varied 1500-fold, whereas 5 alpha-reductase activity varied only 3-fold. Both enzyme activities were highest in amygdaloid, medial preoptic, and medial diencephalic nuclei and lowest in the caudate nucleus. Aromatase activities in the supraoptic nucleus, periventricular area, medial preoptic area-anterior hypothalamus, and lateral hypothalamus were significantly (P less than 0.05) lower in castrated males. Castration did not significantly affect 5 alpha-reductase activity, except for an increase in the basolateral amygdala. The highest concentrations of LHRH were in the infundibular nucleus-median eminence and were 30 times greater than amounts measured in preoptic and medial hypothalamic nuclei. The LHRH contents of the infundibular nucleus-median eminence, ventral medial nucleus, and lateral hypothalamus were significantly lower in castrated males (P less than 0.05). In addition, we observed a significant correlation between aromatase activity and LHRH content in the basal hypothalamus of intact males (r = 0.947; P less than 0.05; n = 8), but not in the preoptic-anterior hypothalamus (r = 0.068; P greater than 0.05; n = 10). No correlation was observed between 5 alpha-reductase activity and LHRH content in either area. These data indicate that castration selectively affects androgen metabolism and LHRH content in discrete regions of the brain of male monkeys and suggest that aromatase and 5 alpha-reductase are regulated differentially in the primate brain.     

Testosterone raises neuropeptide-Y concentration in selected hypothalamic sites and in vitro release from the medial basal hypothalamus of castrated male rats

Although neuropeptide-Y (NPY)-containing neurons are widely distributed in the hypothalamus, castration decreased NPY concentrations only in the median eminence (ME), arcuate nucleus (ARC), and ventromedial nucleus (VMN). We have now examined the effects of testosterone (T) replacement in 2-week castrated male rats on NPY levels in hypothalamic and preoptic area regions and in vitro NPY release in three experiments. In the first experiment we studied the effect of T on NPY concentration in castrated rats. Two-week castrated rats were implanted sc with T-filled or empty Silastic capsules 30 mm in length. Ten days later rats were killed, and NPY levels were measured by RIA in microdissected sites. T implants raised serum T levels to the range found in gonad-intact rats and decreased serum LH levels to the basal range. Further, of the six brain sites examined, significant increases in NPY concentrations occurred selectively in the ME, ARC, and VMN of T-implanted rats. In the second experiment, the ability of T to reverse the effect of castration on NPY levels compared to those in intact (sham) rats was assessed. Again, castration decreased NPY levels in the ME, ARC, and VMN only, and replacement of physiological levels of T restored NPY levels approximately 100%, 127%, and 74% in the ARC, VMN, and ME, respectively. In the third experiment, the effect of castration and T implants (30-mm T capsules for 10 days) to 2-week castrated rats on the in vitro release of NPY from medial basal hypothalamus (MBH) was assessed. Basal NPY release was not significantly changed after castration and T replacement. However, in response to a 30-min pulse of KCl (45 mM) NPY release from the MBH of castrated rats was significantly reduced compared to that in intact and T-replaced castrated rats. These studies show that castration decreases and T replacement restores NPY levels selectively in three hypothalamic sites, viz. ME, ARC, and VMN, and KCl-induced NPY release from the MBH in vitro is decreased after castration and restored by T replacement, thereby suggesting that a local subset of androgen-concentrating neurons may regulate NPY levels and release in a site-specific manner. Further, these results are in line with our emerging view that gonadal steroids modulate neurosecretion not only of LHRH, but also of other functionally linked regulatory peptides.     

Neuropeptide Y levels in microdissected regions of the hypothalamus and in vitro release in response to KCl and prostaglandin E2: effects of castration

Intracerebroventricular administration of neuropeptide Y (NPY) has been shown to modify LH secretion, with the direction of the response dependent on the steroid background. To study further the role of gonadal steroids in the regulation of NPY secretion, the basal and KCl-evoked release of NPY from the medial basal hypothalamus (MBH) of intact and castrated male rats was assessed twice with the use of an in vitro incubation system. In each experiment, the amounts of NPY released in response to a 15-min pulse of KCl (45 mM) were significantly smaller from the MBH of castrated rats than of intact rats (P less than 0.05). Next, to assess the possible effects of prostaglandin E2 (PGE2), the MBH were exposed in a similar manner to two 15-min pulses, 30 min apart, of 0.568 and 56.8 mumol PGE2. Unlike KCl, PGE2 failed to stimulate NPY release from the MBH of either intact or castrated rats. However, a similar 56.8 mumol concentration of PGE2 was effective in stimulating the release of LHRH. We next examined the effects of castration on NPY levels in several microdissected regions of the hypothalamus. Whereas NPY concentrations were unchanged in the medial preoptic area, paraventricular nucleus and dorsomedial nucleus, NPY levels were significantly decreased in the median eminence, arcuate nucleus, and ventromedial nucleus 2 weeks after castration. These studies show that KCl can stimulate NPY release from the MBH in vitro, like that of LHRH, the KCl-induced NPY response is significantly smaller from the MBH of castrated than intact males, castration can significantly reduce the levels of NPY in the median eminence, arcuate nucleus, and ventromedial nucleus, thereby suggesting that testicular secretions may modulate NPY levels and release from the MBH, and because PGE2 stimulated the release of LHRH but not of NPY, separate regulatory neural events may underlie the secretion of these two neuropeptides.     

Differential response of luteinizing hormone-releasing hormone in the basal hypothalamus and the preoptic area following anterior hypothalamic deafferentation and/or castration in male rats.

Serum LH, FSH and LHRH concentrations and the LHRH content in the medial basal hypothalamus (MBH) and the preoptic area (POA) were measured by radioimmunoassay in male rats 33 days after anterior hypothalamic deafferentation (AHD) and/or castration. In castrate rats following AHD, there was a significant decrease in serum LH, FSH, and LHRH concentrations, whereas, in intact rats, serum LH was elevated in AHD over the sham AHD rats. Castration and AHD each caused a significant fall in the LHRH levels in the MBH; the decline was more pronounced in rats undergoing both castration and AHD. In contrast, deafferentation in intact and castrate rats resulted in the accumulation of LHRH activity in the POA. These studies support the suggestion that a) a substantial amount of LHRH normally found in the MBH of intact and castrate male rats originates in the rostral regions and, b) the LHRH-containing neural elements within the MBH have the competence to respond to a loss in the circulating testicular steroids.     

Single cell levels of hypothalamic messenger ribonucleic acid encoding luteinizing hormone-releasing hormone in intact, castrated, and hyperprolactinemic male rats

We have examined the changes that occur in neuronal expression of LHRH mRNA in response to castration and hyperprolactinemia in male rats. Single cell levels of LHRH mRNA were determined by quantitative in situ hybridization histochemistry using an 35S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Nine days postcastration, a 10.4-fold increase in mean plasma LH titers was observed which was associated with significantly increased LHRH mRNA in rostral hypothalamic neuronal cell bodies. Both increases were blocked in rats rendered hyperprolactinemic by the presence of the 7315a PRL-secreting pituitary tumor. The location and number of neurons expressing LHRH mRNA were unchanged, indicating that these differences were attributable to amounts of mRNA expressed per neuron. Experimental differences occurred in LHRH perikarya situated throughout the rostral hypothalamus from the organum vasculosum of the lamina terminalis to the caudal extent of the medial preoptic nucleus. These results suggest that gonadal steroids and PRL are involved, either directly or indirectly, in regulating the biosynthesis of LHRH in the rostral hypothalamus.     

Castration of male rats reduces the capacity of granules isolated from the median eminence to secrete luteinizing hormone-releasing hormone in response to copper

The effect of castration of male rats on the secretory function of median eminence area (MEA) granules containing luteinizing hormone-releasing hormone (LHRH) was examined, using copper as a test substance. LHRH granules, isolated from MEA of sham or castrated rats 1, 2, or 12 weeks postoperatively, were incubated with various concentrations of copper complexed to histidine (CuHis) and the kinetic constants of LHRH release estimated. CuHis-stimulated release of LHRH was found to be a saturable function of the concentration of CuHis. Castration did not alter the apparent Km for CuHis-stimulated release of LHRH; the Km being 3 and 16.9 microM copper for granules obtained from 1-2 weeks and 12 weeks sham-operated rats, respectively, and 3.7-5.6 and 10.2 microM copper for the granules of castrated rats, respectively. Moreover, castration did not alter the fractional amount of LHRH released in response to CuHis. In contrast, castration markedly reduced the actual amount of LHRH released in response to CuHis; the Vmax being 1,933 and 2,942 pg LHRH released/6 min/MEA for 1-2 weeks and 12 weeks sham-operated rats, respectively, and 782 and 757 pg for the castrated rats, respectively. Similarly, the LHRH content of the MEA of castrated rats was lower than that of the shams. These results are suggestive that castration reduces the capacity of the MEA granules to release LHRH in response to a secretion stimulus such as copper and that this is due not to alterations in the biochemical parameters underlying the release process itself but to a reduced level of MEA LHRH available for release.     

Organization of angiotensin II immunoreactive cells and fibers in the rat central nervous system. An immunohistochemical study

The distribution of angiotensin II (AII)-immunoreactive cells and fibers was examined in adult male Sprague-Dawley rats with and without colchicine pretreatment. As seems to be the case for a number of other neuropeptides, AII is preferentially found in brain stem, hypothalamic, and limbic structures involved in the control of homeostatic functions. AII-stained cell bodies were most prominent in magnocellular parts of the paraventricular and supraoptic nuclei, and cells were also found in parvocellular parts of the former. Other hypothalamic nuclei containing cell bodies include the suprachiasmatic nucleus, the medial preoptic area, and perifornical parts of the lateral hypothalamic area. Of considerable interest was robust staining in several of the circumventricular organs, in particular the subfornical organ, where both cells and fibers were found. The results of water deprivation and nephrectomy suggest that this staining does not represent uptake of circulating peptide, but instead, represents AII-containing neural connections. In the thalamus, AII-stained cells were found in the paraventricular nucleus, the central medial nucleus, the nucleus reuniens, and rostral parts of the zona incerta. Two cell groups in the basal telencephalon, in the dorsal part of the bed nucleus of the stria terminalis and in the medial nucleus of the amygdala, lay at either end of an AII-stained pathway coursing through the stria terminalis. In the midbrain, immunoreactive cells were found in the interpeduncular and peripeduncular nuclei, and one pontine cell group was detected in the most lateral part of the lateral parabrachial nucleus. The only AII-stained cells in the medulla were in the nucleus of the solitary tract, near the margin of the area postrema. Fibers were found at all levels of the central nervous system, from the olfactory bulbs to the spinal cord, where terminal fields were observed in the substantia gelatinosa and in the intermediolateral cell column. Longitudinally oriented fibers were present throughout the periventricular fiber system and in the medial forebrain bundle, including its caudal extension in ventrolateral parts of the brain stem. It is suggested that, at many different levels, AII serves as both a hormone and neurotransmitter for fluid balance.     

Close juxtapositions between LHRH immunoreactive neurons and substance P immunoreactive axons in the human diencephalon

LHRH release is induced by substance P (SP) in the rat hypothalamus. Recent immunocytochemical studies indicate that SP-immunoreactive axons synapse on LHRH neurons in the diencephalon of the rat, but this phenomenon has not yet been demonstrated in human. Therefore, in the present study we visualized the SP- and LHRH-immunoreactive (IR) elements in the human diencephalon and evaluated the close juxtapositions between them. The distribution of LHRH- and SP-IR sites were investigated in diencephalic sections of six, postmortem human brains by means of double-labeling immunocytochemistry. The LHRH-containing perikarya were located in the diagonal band of Broca, lamina terminalis cinerea, preopticoseptal, medial preoptic, and infundibular areas of the brain. The SP-IR fibers formed a network in the periventricular zone in the infundibular region, median eminence, and corpus striatum. The SP-IR cell bodies were located mainly in the infundibular region, median eminence, basal part of the periventricular area, dorsomedial subdivision of the ventromedial nucleus, and basal perifornical area of the tuberal region. The juxtapositions between LHRH-IR cell bodies and SP-IR varicosities were detected in the infundibular and periventricular regions. In these sites black, silver-intensified, SP-IR fiber varicosities abutted on brown, DAB-labeled, LHRH-IR cell bodies. Similar structures were detected between the SP-IR fibers and SP-IR perikarya. These findings suggest that the juxtapositions between the SP and LHRH systems may be the morphological basis of SP-controlled LHRH release in the human diencephalon. Moreover, the intimate contacts between SP-IR fiber varicosities and SP-IR cell bodies or axons indicate direct control of SP on the diencephalic SP release.     

The gonadotropin-releasing hormone neuronal system of the male Djungarian hamster: distribution from the olfactory tubercle to the medial basal hypothalamus

The neuroanatomical distribution and morphology of neurons that produce gonadotropin-releasing hormone (GnRH) in the brain of the postpubertal male Djungarian hamster was studied using light microscopic immunocytochemistry. Analysis of every section from the rostral olfactory tubercle to the medial basal hypothalamus indicate 356 +/- 37 immunoreactive GnRH perikarya per brain (mean +/- SE; n = 4 brains). Over 90% of GnRH cell bodies were found in 6 brain regions; the largest number of somata were located in the medial preoptic area followed by the diagonal band, lateral hypothalamus, lateral preoptic area, lateral septum and anterior hypothalamus. Morphologically, two predominant types of GnRH neurons were identified: unipolar GnRH cells with an ovoid soma and only a single distinct process (about 40% of all GnRH neurons), and bipolar cells with a fusiform-shaped perikaryon. Overall and in most brain regions, the ratio of unipolar to bipolar GnRH perikarya was 2:3 or greater. A significant proportion of GnRH neurons had an unusually "thick" process(es) that exited the soma and tapered gradually. GnRH fibers were evident in most sections, forming dense plexuses in the arcuate nucleus-median eminence, the periventricular region of the third ventricle and organum vasculosum of the lamina terminalis. These findings indicate that the Djungarian hamster is similar to other rodent species, especially the white-footed mouse, in the neuroanatomical distribution of GnRH neurons. The present report provides a working atlas for the rostral ventral forebrain of the postpubertal Djungarian hamster.(ABSTRACT TRUNCATED AT 250 WORDS)     

The luteinizing hormone-releasing hormone (LHRH) systems in the rat brain

Immunocytochemical procedures on thick, unembedded sections were used to visualize the neurons and their processes that contain LHRH-immunoreactive material in the rat central nervous system (CNS). In animals pretreated with colchicine (75 micrograms, intraventricularly), cell bodies could be observed as far anterior as the olfactory bulb and posterior to the retrochiasmatic area of the basal hypothalamus. Several new observations for the rat were made in this study, including LHRH neurons in the accessory olfactory bulb and other olfactory-related structures, and in the anterior hippocampus and the induseum griseum. As in studies from other laboratories, we observed many LHRH cells in the periventricular medial preoptic area, diagonal band of Broca and septal nuclei, and fewer positive cells in the anterior hypothalamic area and the region of the supraoptic commissure. The LHRH fibers from all of these cells are widely dispersed in the CNS. In addition to the dense innervation of the median eminence, positive fibers are found innervating other circumventricular organs, coursing close to the ependymal wall of the ventricular system or in close association with cerebral arteries and areas of the pia mater and subarachnoid space. LHRH fibers may also innervate neurons in several regions of the CNS. A novel projection of LHRH fibers for the rat was found originating from supracallosal neurons and coursing through both cingulate and neocortex. The possible distribution of efferents from each LHRH cell group is discussed.     

Testosterone raises LHRH levels exclusively in the median eminence of castrated rats

Although LHRH is widely distributed in the diencephalon, previous studies show that testosterone (T) treatment for 72-96 h of castrated male rats raised LHRH levels only in the medial basal hypothalamus. In the present study, LHRH concentrations were analyzed in microdissected brain regions shown to contain LHRH perikarya and their projections to identify the discrete regions which may display this T-dependent accumulation of luteinizing hormone-releasing hormone (LHRH) and to study the temporal sequence of LHRH changes in those regions. Rats were killed at 12-hour intervals from 60 to 96 h after subcutaneous implantation of Silastic capsules containing T. Results showed that the physiological range of serum T levels attained by these implants suppressed LH release at all times, however, there was no immediate effect on LHRH concentrations in any region. In fact, out of the 9 regions in the preoptic-tuberal pathway examined, 8 regions displayed no change in LHRH concentrations at any time. On the other hand, for up to 84 h, LHRH concentrations in the median eminence region remained unchanged; an additional 12 h of T exposure significantly raised LHRH levels. These results suggest that this T-dependent accumulation of LHRH may arise either by de novo synthesis or by increase in the rate of processing of the precursor LHRH protein into the immunoreactive form within nerve terminals in the median eminence.     

Inhibitory effect of hypothalamic medial preoptic area somatostatin on growth hormone-releasing factor in the rat

Possible inhibitory effects of somatostatin (SRIF) on GRF were studied by assessing spontaneous GH secretion and GRF content and release in adult male rats depleted of hypothalamic SRIF by anterolateral hypothalamic deafferentation (AHD) or electrolytic lesions in the medial preoptic area (MPO). Plasma GH levels were measured 7 days postoperatively every 20 min in conscious animals with indwelling iv cannulae. Median eminence SRIF was markedly reduced 8 days postoperatively in both AHD and MPO rats, as determined by immunohistochemistry and RIA (P less than 0.01). Although GRF immunoreactivity in the median eminence of AHD and MPO animals appeared well preserved immunocytochemically, hypothalamic GRF content by RIA was significantly decreased at 8 days (P less than 0.01). Spontaneous GH secretion was pulsatile in sham-operated animals. In contrast, basal GH levels in AHD and MPO animals were markedly elevated (P less than 0.01), and secretory pulses were absent. Intravenous injection of specific anti-GRF serum into MPO animals decreased the elevated plasma GH levels (P less than 0.01), indicating increased hypothalamic GRF secretion. GRF release from hypothalamic median eminence-arcuate nucleus complexes in vitro was significantly greater in AHD and MPO animals than in control animals 4 and 8 days postoperatively in response to 30 mM K+ (P less than 0.01), but not under basal conditions. These results suggest that hypothalamic medial preoptic area somatostatinergic neurons play a tonic inhibitory role in the regulation of GRF release and that GH hypersecretion observed after MPO and AHD is attributable to changes in both SRIF and GRF.     

Sex difference in the effect of mating on c-fos expression in luteinizing hormone-releasing hormone neurons of the ferret forebrain.

The pulsatile secretion of LH was previously found to rise in female ferrets after receipt of an intromission, whereas in males that achieved an intromission, both LH and testosterone secretion were either reduced or unchanged. We sought to determine whether this sexually dimorphic pattern of LH secretion reflects a sex difference in the effect of mating on the activity of forebrain neurons that secrete LHRH. Immunocytochemical methods were used to localize the nuclear protein product of the immediate early gene, c-fos, as an index of increased neuronal activity after mating. Nuclear FOS immunoreactivity (FOS-IR) was monitored in LHRH-IR neurons as well as other non-LHRH forebrain neurons. In confirmation of previous reports, LHRH-IR perikarya in ferrets of both sexes were located medially along the base of the brain at rostral, medial, and caudal levels of the preoptic-hypothalamic continuum. In each of these regions a significantly higher percentage of LHRH-IR neurons was colabeled with nuclear FOS-IR in mated than in unpaired females. By contrast, an equivalent low percentage of LHRH-IR neurons was colabeled with FOS-IR in mated and unpaired male ferrets. Significantly more FOS-IR neurons (not colabeled with LHRH) were detected in the bed nucleus of the stria terminalis, the medial preoptic area, the dorsal-medial hypothalamus, and the medial amygdala (MA) of mated vs. unpaired females. By contrast, mating significantly augmented FOS-IR only in the MA of male ferrets. The results suggest that the sexually dimorphic pattern of LH secretion that occurs in ferrets after mating reflects a selective activation of LHRH neurons in the female forebrain. This sex-specific increase in the responsiveness of LHRH neurons to mating may depend on input from a limbic circuit which includes the medial amygdala, bed nucleus of the stria terminalis, and medial preoptic area.     

Opiate-induced hypersensitivity to testosterone feedback: pituitary involvement

We have examined the mode of morphine's (M) action to increase the sensitivity of castrated male rats to the inhibitory feedback action of testosterone (T) on LH release. In castrated rats, sc implantation of M pellets or 5-mm long T-filled capsules (T5) failed to suppress LH release, but a combination of M and T5 drastically decreased serum LH levels. Likewise, while treatment with a higher dose of T (30-mm long implant, sc) suppressed LH release, combined treatment with M and T30 produced a further suppression of LH levels. We have now assessed the in vitro release rate of LHRH from the medial basal hypothalamus-preoptic area of castrated rats treated with M and/or T as well as the in vivo pituitary LH response to LHRH challenge in similarly treated rats. Interestingly, the in vitro basal and naloxone-induced LHRH release from the medial basal hypothalamus-preoptic area of the six groups of rats was similar, regardless of whether LH levels were in the high castrate or low basal range. On the other hand, M treatment greatly attenuated LH release in vivo in response to LHRH challenge (10pmol-1pmol) [corrected] in T-treated rats. In fact, LH increments in response to 1pmol [corrected] LHRH, seen in control, T5, and T30 groups, were abolished by additional M treatment of T-treated rats. This in vitro assessment of LHRH release suggests that the drastic decrease in LH release in T-plus M-treated rats may not be due to impaired LHRH release, but, rather, be due in part to reduced pituitary responsiveness to intermittent endogenous LHRH signals. The reduced pituitary responsiveness to LHRH in T-plus M-treated rats may be a consequence of either a direct pituitary effect of opiates in conjunction with T or augmented action of hypothalamic neurohumoral agents which may inhibit LH release on their own or antagonize the LH-releasing action of LHRH at the level of pituitary gonadotrophs.     

Circadian periodicities of serum androgens, progesterone, gonadotropins and luteinizing hormone-releasing hormone in male rats: the effects of hypothalamic deafferentation, castration and adrenalectomy.

Serum testosterone (T), dihydrotestosterone (DHT), progesterone (P), LH, FSH and luteinizing hormone-releasing hormone (LHRH) levels and the LHRH content in the medial basal hypothalamus (MBH) and preoptic area (POA) were estimated during a 24-h period. Circadian rhythms, temporally unrelated to each other, were evident in serum FSH and LHRH and the MBH LHRH content; serum LH fluctuated randomly. In addition, serum T and DHT demonstrated a parallel circadian pattern while serum T and P appeared inversely related. Serum P rhythm persisted in long-term castrate males. Without adversely affecting serum LH, anterior hypothalamic deafferentation abolished both serum T and P rhythms, whereas adrenalectomy obliterated only the serum T periodicity. These studies show that the POA-MBH complex and the adrenals play important roles in the circadian regulation of testicular secretions.     

Interactions of pinealectomy and short-photoperiod exposure on the neuroendocrine axis of the male Syrian hamster

The effect of pineal gland removal on neuroendocrine function of male Syrian hamsters housed under long (14 h light:10 h dark) or short (5 h light 19 h dark) photoperiod conditions was tested. In sham-operated, but not in pinealectomized, animals, exposure to the short photoperiod resulted in a significant reduction in testicular weight. Median eminence (ME), medial basal hypothalamus (MBH), and medial preoptic-suprachiasmatic (MPOA-SCN) norepinephrine (NE) turnover was significantly reduced in 5 L:19 D sham-operated animals as compared to 14 L:10 D sham-operated or 14 L:10 D pinealectomized controls. The effects of short photoperiod on ME and MPOA-SCN NE turnover were reversed by pinealectomy, but reductions in MBH NE turnover were not dependent on the presence of the pineal gland. Pineal-dependent decreases in MBH and increases in MPOA-SCN dopamine turnover were also observed after transfer of hamsters from long to short photoperiods. Both ME and MBH luteinizing hormone-releasing hormone (LHRH) levels were increased after short-photoperiod exposure, but pineal removal prevented these increases of LHRH levels only in the MBH of the 5 L:19 D hamsters. Levels of serotonin or its metabolite, 5-hydroxyindole-acetic acid, were not affected by pinealectomy and/or short-photoperiod exposure. We conclude that short-photoperiod-induced gonadal atrophy in the Syrian hamster is associated with pineal-dependent and pineal-independent changes in hypothalamic neurotransmitter turnover and hypothalamic LHRH content.     

Effects of testosterone implants and hypothalamic lesions on luteinizing hormone regulation in the castrated male rat.

The effects of intrahypothalamic and subcutaneous implants of testosterone (T) and those of hypothalamic lesions on resting levels of circulating LH and pituitary responsiveness to exogenous LHRH were studied in castrated male rats to elucidate hypothalamic and pituitary regulation of LH secretion. Two hundred mug implants of testosterone propionate (TP) in the median eminence region suppressed plasma LH titers before evidence of direct inhibition of pituitary function (as indicated by testing with LHRH) was found. Such implants release appreciable amounts of T into the peripheral circulation in the immediate post-operative period, and SC Silastic (constant release) capsules containing T have similar effects. The findings suggest that, regardless of the site of implant, the initial negative feedback inhibition of LH by T is not dependent on direct action at the pituitary levels but rather appears to be a hypothalamic effect. In the days following exposure to hypothalamic or peripheral implantation of T, however, a progressively developing decline in the response to exogenous LHRH was observed. In order to determine whether this effect results from suppression of endogenous LHRH release, the median eminence-arcuate region was destroyed to remove the source of LHRH. In these animals, the suppression of plasma LH was evident on the first day after the lesion, but pituitary responsiveness to LHRH was unaffected until after one week. When Sialastic capsules were implanted SC into lesioned animals, a more rapid (less than 1 week) inhibition of pituitary responsivity ensued. Suprachiasmatic lesions did not affect basal LH secretion or pituitary responses to LHRH. The data provide evidence for a dual feedback action of T on LH in castrated male rats: an initial inhibitory effect presumably due to hypothalamic inhibition (commencing at around 6h after hypothalamic of SC implantation of T), and a subsequent suppression of pituitary responisveness (after one day) presumably due to direct action of T on the pituitary. In addition to these phenomena, findings in rats bearing median eminence-arcurate lesions suggest that the removal of endogenous LHRH by itself leads to an eventual decline in pituitary responsiveness (greater than one week postoperatively).     

Evidence that hypothalamic neuropeptide Y secretion decreases in aged male rats: implications for reproductive aging

The decrease in circulating testosterone and LH titer that occurs in aged male rats may in part be a consequence of decreased excitatory neurochemical signals that promote the episodic discharge of LHRH from the hypothalamus. In view of evidence that neuropeptide Y (NPY) stimulates the release and potentiates the action of LHRH on LH secretion, the present studies investigated age-related changes in the concentrations of NPY in individual hypothalamic nuclei and in the ability of hypothalamic tissues to release NPY in vitro. Compared with tissues of 2.5-month-old male rats, medial basal hypothalamic tissues of 13-month-old rats released significantly less NPY in response to K+ depolarization. In contrast, K+-evoked LHRH release from the same tissues was unimpaired. Aged male rats also exhibited markedly reduced concentrations of NPY in the median eminence and in the arcuate, medial preoptic, suprachiasmatic, paraventricular, dorsomedial, and ventromedial hypothalamic nuclei, which are sites of NPY perikarya and nerve terminal networks. These neurochemical changes occurred in association with decreased serum testosterone and LH levels. These findings demonstrate a widespread age-related decline in NPY levels and release in the hypothalamus, which may be responsible for the reduction in testosterone secretion and may contribute to the decline in reproductive function in aged male rats.     

Alterations in brain atrial natriuretic polypeptide levels in hypophysectomized rats.

Twelve days after hypophysectomy depleted atrial natriuretic polypeptide (ANP) concentrations were measured in the plasma and in 8 of 18 microdissected brain nuclei of rats. Reduced ANP levels were found in brain structures (subfornical organ, organum vasculosum laminae terminalis, preoptic and hypothalamic periventricular nuclei, paraventricular nucleus, lateral hypothalamic area), which are directly involved in the central regulations of salt and fluid homeostasis, as well as in the medial amygdaloid nucleus and the locus ceruleus. ANP concentrations in the median eminence, medial preoptic and arcuate nuclei did not alter by hypophysectomy. Elevated ANP concentrations were measured only in the supraoptic nucleus of hypophysectomized rats.